Anti-C5 alpha antibodies

ABSTRACT

The present invention refers to recombinant antibodies of human origin specific for the C5 component of the activated complement and characterised by the ability to inhibit the conversion of the C5 alpha chain to C5 a  and C5 b . Moreover the present invention refers to the nucleotide sequences coding for such antibodies and to the therapeutic use of both polypeptide and nucleotide sequences, in particular for the therapy of diseases involving tissue damage deriving from uncontrolled activation of the complement system.

This application is a U.S. national stage application under 35 U.S.C.§371 of PCT/EP2003/007487, filed Jul. 10, 2003, which claims priorityfrom Italian application number MI2002A001527, filed Jul. 11, 2002.

This application includes a “Sequence Listing,” which is provided as anelectronic document on a compact disc (CD-R). This compact disc containsthe file “Substitute Sequence Listing.txt” (24 kilobytes, created onSep. 24, 2010), which is hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION AND BACKGROUND OF THE ART

Activation of the complement system (C system) represents an importantmechanism in the immune defence. At the same time it represents adouble-edged weapon because on the one hand guarantees protection of thehost but on the other is capable of damaging tissues where complement isactivated by several pathological circumstances. The increasedsusceptibility to bacterial infections and autoimmune disorders observedin patients with inherited deficiencies of the C system clearlydemonstrates the particular importance of this system in host protectionagainst infectious agents and in the clearance of immunocomplexes.

These protective functions result from complement activation in cascadefashion that generates biologically active products. Some of those, suchas C1q, C3b and C3bi, opsonize the infectious agents enabling theirdisposal. Instead others, such as C5a and C5b67, have the function torecruit phagocytic cells at the site of inflammation or lyse sensitivetargets as in the case of the membrane attack complex (MAC).Unfortunately these molecules, once they are produced, are not able todiscriminate between endogenous and exogenous targets, and would provokeserious damage to tissues and cells if those were not protected bypotent membrane or extracellular inhibitors acting at various levels inthe complement activation cascade. However, the action of inhibitors isoverwhelmed in presence of a massive activation of the C system, inserious infectious diseases or autoimmune disorders, and the activatedcomplement causes tissue and cell destruction.

The C5a fragment and the C terminal complex (TCC) are among the productsinvolved in tissue destruction in several inflammatory processes. Abovenormal levels of these activation products can be found in synovialfluid of rheumatoid arthritis patients and in cerebrospinal fluid ofpatients with several diseases of the central nervous system. ElevatedC5a levels have been found also in polytraumatised patients as well asin patients with damage from myocardial ischaemia and re-perfusion.

Therefore the role of C5a in development of these diseases is nowrecognised.

This is demonstrated by the signs of pulmonary stress, hypotension andleukopenia shown by animals that have received intravenous injection ofthis anaphylatoxin. Moreover bronchial instillation of C5a is capable ofinducing strong inflammatory reactions in rabbit lung.

TCC is generated from the C5b fragment released by enzymatic cleavage ofC5 by the action of C5 convertase. Recently it has been demonstratedthat TCC induces inflammation because of tissue damage deriving from itslytic activity and of numerous non-cytotoxic effects on phagocytes andother cell types.

TCC has been identified in several tissues in diverse pathologicalconditions, including rheumatoid arthritis, glomerulopathies multiplesclerosis, demyelinating peripheral neuropathies, atherosclerosis andmyocardial infarction. The development of experimental animal models ofthese diseases with selective deficiencies in late C components hasfurther contributed to define the role of these components indevelopment of tissue damage.

Because of the fundamental role played by C5a and TCC in promotingchronic inflammation and tissue damage, several attempts have been madeto neutralise the late components of the C complex as therapeuticstrategy to prevent these complications in diseases associated with C5activation. This molecule turns out to be an ideal therapeutic target,since its neutralisation inhibits the late sequence of activation eventsof the cascade, without interfering with the opsonizing activity of theearly components of this cascade. Mouse monoclonal antibodies specificfor human, mouse and rat C5 and capable of inhibiting the production ofC5a and of the membrane attack complex (MAC) are already commerciallyavailable. Anti-C5 antibodies have been successfully used in mice toprevent the development of collagen-induced arthritis and to improve theclinical curse of glomerulonephritis, and in rats to reduce myocardialischemia and re-perfusion.

In the last years two single chain antibodies (single chain antibody orsingle chain fragment variable, scFv) have been produced that are bothdescribed in patent application WO 95/29697. These antibodies are ableto penetrate tissues more rapidly than the whole antibody. The firstsingle chain antibody obtained by assembling variable regions of a mouseantibody for C5 retains the ability of the original antibody to inhibitassembly of the MAC and partially block C5a production (Evans, M. J etal, 1995, Mol. Immunol. 32:1183). Moreover, this antibody is able toprevent C5b-9 deposits in the heart re-perfused with human plasma or inheart insufficiency. The second scFv is a humanised mouse antibodyanti-C5, that is obtained by inserting murine CDR regions(complementarity determining region) in the structure of the variableregion of human light and heavy chains. This antibody (Thomas, T. C. etal., 1996, Mol. Immunol. 33:1389) is able to inhibit C5a and C5b-9formation although the recognised epitope mapped around amino acids860-865 of the C5 molecule and corresponding to peptide KSSKC [SEQ IDNO: 36], turns out to be far from the C5 convertase cleavage site.Moreover subsequent studies (Fitch, J. C. et al., 1999, Circulation100:2499) have demonstrated that this antibody is able to inhibitcomplement hemolytic activity, to attenuate myocardial damage, cognitivedamage and postoperative haemorrhage in a group of patients withcardiopulmonary bypass.

SUMMARY

The main aspect of the invention refers to an antibody of human originspecific for the C5 component of activated complement and characterisedby the fact that it inhibits cleavage of the C5 alpha chain into C5a andC5b. In particular, the antibody is recombinant and recognises anepitope comprising the proteolytic site for the C5 convertase on thealpha chain of the C5 complement component.

According to a preferred embodiment, the recombinant antibody iscomposed of a single chain (scFv) comprising a variable region of thelight chain covalently bound to a variable region of the heavy chainand, according to an even more preferred aspect is composed of orcomprises at least one of amino acid sequences selected among SEQ ID NO:2, 4, 6 or proteins having at least 95% homology to such polypeptides.

In accordance with a further aspect, the invention includes the isolatednucleotide sequences encoding antibodies specific for C5 component ofactivated complement. These antibodies are characterised by the factthat they inhibit cleavage of the C5 alpha chain into C5a and C5b, andin particular the sequences chosen between: SEQ ID NO: 1 or 3 or 5 andthe vectors containing these sequences. In accordance with a furtheraspect, the invention refers to the therapeutic use of antibodies andnucleotide sequences for preparation of drugs that prevent and treatdiseases involving uncontrolled activation of the complement system, inparticular: rheumatoid, arthritis, glomerulonephrits, multiplesclerosis, demyelinating peripheral neuropathies, atherosclerosis.

DESCRIPTION OF THE FIGURES

FIG. 1. Assessment of the ability of anti-C5 clones scFv Ts-a12 andTs-a12/22 to inhibit formation of C5a fragment by means of an ELISAassay (panel A) and an haemolytic assay (panel B).

Panel A) The ELISA assay measures the amount of C5a fragment released inthe supernatant after enzymatic cleavage of C5, using antibodies anti-C5as described in example 6. Incubation of C5 with antibodies Ts-a12 andTs-a12/22 of the invention inhibits formation of the C5a fragment. Sheeperythrocytes sensitised with antibodies and coated until the C3b(EAC1-3b) complement step were added to the mixture of TS-A12 orTS-A12/22 and C5, and the mixture was further incubated for 30 minutesat 37° C. (panel B). As shown in FIGS. 1A and B, TS-A12 and TS-A12/22antibodies inhibit C5 cleavage by C5 convertase and therefore inhibitformation of C5a (Panel A) and of TCC (Panel B).

Symbols: -▴-: Ts-A12/22; -▪-: unrelated scFv; -⋄-: TS-A12.

FIG. 2. Immunoblotting to identify the C5 chain recognised by scFvTSA-12/22.

The alpha and beta chains of C5 were electrophoretically separated bymeans of SDS-PAGE in 10% polyacrylamide and then transferred ontonitrocellulose. The immunoblot (lanes 1 and 2) was developed withTSA-12/22 antibody and revealed by incubation with anti-SV5 (SV5 tag)monoclonal antibody followed by incubation with a goat anti-mouse IgGantibody labeled with alkaline phosphatase. Lane 1: 100 ng of C5 alphachain; lane 2: 100 ng of beta chain; lane 3: mixture of the two chains.Immunoblot in lane 3, used as positive control, was developed with abiotin conjugated goat anti-human C5 antibody that recognises both alphaand beta chains and was revealed with alkaline phosphatase labeledavidin. It can be seen that scFv TSA-12/22 recognises the alpha chain ofC5 in lane 1, but not the beta chain in lane 2.

FIG. 3. Inhibition of the binding of C5 and scFv of the invention bypeptide P5A-18 (KDMQLGRLHMKTLLPVSK) (SEQ ID NO:15) comprising the C5convertase cleavage site.

Mixtures of scFv TSA-12/22 (1 μg/ml) containing 200, 400 and 800 ng ofP5A-18 peptide, with sequence KDMQLGRLHMKTLLPVSK [SEQ ID NO:15],comprising the C5 convertase cleavage site, or 800 ng of unrelatedpeptide CS5, derived from fibronectin (GEEIQIGHIPREDVDYHLYP SEQ ID No.16 of sequence listing) or 3.5 μg of C5a fragment or saline solution(control) were incubated as described in example 8. Binding of theantibody, pre-incubated in different conditions, was assessed byimmuno-enzymatic assay using C5 on solid phase. Inhibition by the P5A-18peptide of the binding between scFv of the invention and C5 isdose-dependent, ranging between 45% to 90% for peptide concentrations of200 and 800 ng, respectively. These values correspond to a Ki of 1 μMfor the P5A-18 peptide. No inhibition is observed using C5A or theunrelated peptide.

FIG. 4. Inhibition of hemolytic activity of C5 by the TS-A12/22antibody.

Values from spectrophotometric reading performed at 412 nm to measureinhibition of lysis of sheep erythrocytes coated with EAC1-3b, throughthe classical complement activation pathway (A Panel); or of rabbiterythrocytes in order to measure the alternative pathway of complementactivation (B Panel). Increasing amounts of C5 were mixed with 600 ng ofthe scFv antibody of the invention with an unrelated scFv, or with GVBSin which the scFv of the invention were solubilised, as described inexample 9. After incubation with serum lacking in C5, obtained from apatient with selective deficiency of this complement component, thepercentage of hemolysis was measured compared to the 100% value obtainedby lysing erythrocytes in an equal volume of distilled H₂O and a blankobtained by resuspending eythrocytes in GBVS.

Symbols: -⋄-: TS-A12/22; -▪-: unrelated scFv; -▴-: GVBS.

FIG. 5. Inhibition of C5 hemolytic activity in mammalian serum by theTS-A12/22 antibody.

Measurements were performed as described in example 8, using sera fromhuman (panel A), rat (panel B), rabbit (panel C), mouse (panel D).

Symbols: -▴-: GVBS TS-A12/22; -▪-: unrelated scFv; -⋄-: TS-A12/22.

FIG. 6. Inhibition of intra-articular migration of PolymorphonuclearLeukocytes (PMN) by the TS-A12/22 antibody in rat models ofantigen-induced arthritis.

Polymorphonuclear Leukocytes (PMN), isolated from intra-articular washout of rats with arthritis induced by instillation of BSA (mBSA), wereincubated with antibody TS-A12/22 or with an unrelated antibody, asdescribed in example 10. The number of PMN in the joint treated with theantibody of the invention appears drastically reduced.

Saline solution: control without induced arthritis; mBSA: BSA-inducedarthritis, untreated; unrelated: BSA-induced arthritis, treated withunrelated scFv antibody; Ts-a12/22: BSA-induced arthritis, treated withthe antibody of the invention.

FIG. 7. Immunofluorescence analysis of synovial membrane from rats withantigen-induced arthritis.

Histological sections of joints from rats treated with intra-articularinjection of saline (A), BSA (B), BSA with TS-A12/22 (C) and BSA withthe unrelated antibody (D) were analysed by immunofluorescence for thepresence of complement components C3 and C9, as described in example 10.It is emphasised that the treatment with TS-A12/22 does not affect thedeposition of C3 but significantly reduces that of C9, thus confirmingthe inhibition of C5.

FIG. 8. Schematic representation of minibodies prepared from ScFvsequences including human, mouse and rat constant regions.

FIG. 9. Inhibition of the classical complement activation pathway.

Symbols: -▴-: GVBS; -▪-: unrelated scFv; -⋄-: TS-A12/22-CH2CH3(minibody); -▪-TS-A12/22 (scFv).

FIG. 10. Inhibition of intra-articular migration of PolymorphonuclearLeukocytes (PMN) by the TS-A12/22-CH2CH3 antibody (minibody) in ratmodels of antigen-induced arthritis.

From left: bar 1: control (mBSA); bar 2: mBSA+TS-A12/22-CH2CH3(minibody) injected at time 0; bar 3: mBSA+TS-A12/22-CH2CH3 (minibody)injected at 6 hours from induction of arthritis.

FIG. 11. Reduction of the joint swelling.

The antibody, under the form of minibody, was injected in the model ofmBSA-induced arthritis in rat at time 0 and 6 days after BSA treatment.Percentage values on the ordinate have been obtained with respect to thebasal diameter of the joint prior to induction of arthritis. Days areindicated on the abscissa.

From left: bar 1: control (mBSA); bar 2: mBSA+TS-A12/22-CH2CH3(minibody) injected at time 0 from induction of arthritis; bar 3:mBSA+TS-A12/22-CH2CH3 (minibody) injected at 6 hours from induction ofarthritis.

DETAILED DESCRIPTION OF THE INVENTION

The main object of the present invention relates to an antibody of humanorigin which has specificity for the C5 component of the complementsystem and is characterised by the fact of inhibiting the cleavage of C5alpha chain, also termed activated C5, into C5a and C5b fragments. Thiscleavage occurs as result of complement activation occurring by knownmechanisms.

Following Complement activation, a C5 convertase is produced whichcleaves the alpha chain of factor C5 generating a fragment ofapproximately 70 amino acids (aa), known as C5a, and a C-terminalfragment of 925 aa, C5b. The products resulting from C5 activation, C5aand C5b, are biologically active. In particular, C5a has chemotacticactivity toward polymorphonuclear leukocytes and monocytes, whereas theC5b fragment contributes to formation of the terminal complement complex(TCC).

The antibody is preferably recombinant.

The antibody of the invention is of human origin, i.e. it is entirelyderived from an antibody repertoire obtained from human lymphocytes.These antibodies have both framework and antigen complementary regions(CDR) of human origin, unlike humanised antibodies where only theframework is of human origin, while the CDR are of murine origin.

Herein, by recombinant antibody it is meant an antibody composed of atleast one variable region derived from the heavy or light chain of animmunoglobulin and produced by means of genetic engineering techniquesfrom the nucleotide sequences coding the characterising regions of theantibody. The recombinant antibody is produced in a host organism whichis usually a bacteria, a yeast, or a higher eukaryotic cell (of plant oranimal origin). The technique of genetic engineering allows to produceentirely human antibodies or to select a format different from naturalantibodies. On the contrary, antibodies produced by the classicalhybridoma technology described by Milstein et al., can be only frommouse or rat and have the typical Y shaped format of four-chainantibodies. The four chains, identical pair-wise, comprise a heavy and alight chain, each consisting of a constant and variable region, asdescribed by Rathburn, G. et al. (1989) in Immunoglobulin genes,Academic Press, New York.

According to a preferred embodiment, the anti-C5 recombinant antibody ofthe invention is characterised by the fact of recognising an epitope onthe alpha chain of the C5 component, which comprises the cleavage regionof C5 convertase. The binding specificity of the antibody of theinvention for the epitope comprising the cleavage site for C5convertase, which is positioned between glycine 733 and arginine 734 ofhuman C5 according to the SwissProt Data Base numbering for human C5(SEQ ID P01031), can be verified in vitro. For instance, it can beverified by means of an ELISA competition assay on C5 that makes use ofa synthetic peptide, as for example the peptide KDMQLGR↓LHMKTLLPVSK (SEQID NO:15) (where the arrow indicates the proteolytic cleavage site),corresponding to the region 727-744 of the human mature protein.According to this aspect, the antibody is preferably characterised bythe ability of recognising a region with at least 80%, preferably atleast 90% 95% homology to the peptide KDMQLGR↓LHMKTLLPVSK (correspondingto the SEQ ID NO:15). More preferably it recognises an epitope of atleast 6-10 amino acids composed of 1-5 amino acids upstream and 1-5amino acids downstream the peptide bond cleaved by the enzyme C5convertase, preferably the epitope is LGRLHM (SEQ ID NO:37). The regionsurrounding the cleavage site is highly conserved across severalmammalian species, therefore the antibody of the invention recogniseswith very similar binding efficiency the C5 molecule of rat, mouse,rabbit etc. Moreover the antibody has in each animal species the sameeffect to block the conversion of activated C5 into its active fragmentsC5a+C5b.

The recombinant antibody of the invention is preferably in a singlechain form (scFv), even more preferably it corresponds to SEQ ID NO:6,and comprises the variable region of the light chain covalently joinedto the variable region of the heavy chain, either directly or via anamino acid sequence termed linker. Herein by scFv it is meant a singlepolypeptide chain antibody, composed of the variable region of the lightchain joined to the variable region of the heavy chain either directlyor via synthetic linker. Linkers composed of non-natural amino acidsequences (synthetic) are known in the art and are described forinstance in (1999): Combinatorial Chemistry and Technology: Principles,Methods, and application, Marcel Dekker Inc, NY USA. The syntheticlinker is preferably the nucleotide sequence SEQ ID NO:13 of theSequence Listing.

In the scFv antibody of the invention, VH and VL chains are preferablyin the order VL-VH from the N— to the C-terminus of the polypeptidechain. This format, in which the N-terminal consists of VL chain,confers greater hydrophilicity to the whole protein expressed as suchand to fusion proteins comprising both VL and VH regions. Furthermore itmakes possible the upstream or downstream insertion of peptide sequencestermed “tag” or “flag”, which do not alter the binding characteristicsbut are used for instance to facilitate the immunological detection ofthe antibody, or its production or purification. For the purpose of thepresent invention, the antibody may also consist of the format (N—Cterminus) VH-VL, or it may contain only one of the two variable chains,preferably the VH chain corresponding to SEQ ID NO:4, also inassociation with different VL chains, also independently of theirspecificity, as for instance selectable through interaction of the VHchain corresponding to SEQ ID NO:4 with “collections of molecularrepertoires”.

In the antibody of the invention, the VL chain has a sequence preferablycorresponding to SEQ ID NO:2 that could be covalently linked to a VHchain which preferably corresponds to SEQ ID NO:4.

In the scFv antibody, preferably at least the VH chain has the aboveindicated anti-C5 specificity and preferably corresponds to SEQ ID NO:4of the Sequence Listing or to the isotypic variants or conservativemutations of this sequence. Therefore, according to this aspect, theinvention comprises all polypeptides comprising a region having at least95% homology, preferably 98% or 99% homology with the amino acidsequence of the VH region, preferably VH3 corresponding to SEQ ID NO:4.A particularly preferred embodiment of this scFv is represented by theantibody having amino acid sequence corresponding to SEQ ID NO:6, whichcorresponds to sequence 4 and 2 joined by a linker peptide to a sequencecorresponding to SEQ ID NO:14. In accordance with a particularlypreferred embodiment the scFv antibody corresponding to the SEQ ID NO:6,has an equilibrium constant for the antigen higher than 1×10⁷,preferably higher than 1×10⁸.

In a preferred embodiment, the nucleotide sequence of scFv antibody isused to engineer expression cassettes for recombinant antibodiescomprising the constant region of immunoglobulin heavy chains,preferably IgG, even more preferably of human, mouse or rat origin. Evenmore preferably these constructs comprise CH2 and CH3 regions eitherindividually or in association. The expression cassettes made accordingto this procedure are cloned in suitable expression vectors which areused for transfection of eukaryotic cells, preferably of mammalianorigin, as for instance HEK293, CHO, COS-1 BHK, myeloma cells or othercells suitable for expression of these protein products. According to aparticularly preferred embodiment, the scFv antibody corresponding toSEQ ID NO:5 is produced in recombinant form with rat CH2 and CH3sequences. In this form, the recombinant antibody dimerizes and adimeric scFv TSA22-12 represents a particularly preferred embodiment ofthe invention.

Anyway are enclosed in this scope of the present invention theamino-acid sequences obtained by mutation of the sequences contained inthe annexed Sequence Listing, as long as these mutations do not alterthe described anti-C5 antibody specificity. The mutation can be“conservative”, when it is based on an amino-acid with similarstructural or chemical characteristics with respect to polarity, charge,solubility, hydrophobicity, hydrophilicity or it is based on theamphipathic nature of the amino-acid residues involved. For instancegroups of amino acids sharing similar characteristics of polarity arecomposed by non-polar (hydrophobic) aa which include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan and methionine;non-polar or neutral amino acids that include: glycine, serine,threonine, cysteine, tyrosine, asparagine and glutamine; positivelycharged (basic) amino acids that include: arginine, lysine andhistidine; and the group of negatively charged (acidic) amino acids thatcomprises: aspartic acid and glutamic acid. Mutations also can beproduced randomly, for instance using DNA polymerases known to be “errorprone”. Therefore, according to a further aspect, this inventioncomprises recombinant antibodies generated by mutagenesis of nucleotidesequences SEQ ID NO:3 and 5 corresponding to sequences coding the VHregion and for antibody in the scFv form. In accordance with thisaspect, the invention therefore includes a procedure to generateantibodies with specificity for the C5 component of activated complementwhich may have or not have the ability to block cleavage of C5 alpha inits biologically active components. This procedure essentially includesthe use of any one of the sequences SEQ ID NO:4 or SEQ ID NO:6 orpreferably the random or site directed mutagenesis of the nucleotidesequences encoding for SEQ ID NO:4 and NO:6. Therefore this procedurepreferably includes mutagenesis of the nucleotide sequences SEQ ID NO:3and SEQ ID NO:5.

Within the preferred format of the antibody, consisting of variableregions of antibody light or heavy chain fused in a single scFv chainvia a protein linker, the light chain is more preferably the lambdachain, and in particular the Vλ3/V2-14 chain, or the kappa chain,preferably the Vκ4/DPK24. The heavy chain is the VH3 chain, inparticular the VH3/V-48, as defined in the Vbase index. According tothis aspect the invention comprises all the antibodies derived bymutagenesis of the nucleotide sequence coding the VH chain or the scFvof the invention. These antibodies are characterized by the fact thatthey retain specificity for the C5 component of the complement systemand of inhibiting cleavage of the C5 alpha chain, also termed activatedC5, into C5a and C5b fragments.

It is known to the skilled person that the antigen binding specificityof an antibody is mainly determined by the CDR regions (ComplementarityDetermining Region) which are defined as hyper-variable regions of theantibody. Three hyper-variable regions for each variable region exist inboth the heavy and the light chain. However, not unanimously acceptedare the precise boundaries of the less variable or “framework” regionswithin which the CDR are comprised. Indeed two different classificationsexist. The first is based on “sequence variability” (Kabat et al.),while the second is based on “structural variability” (Chothia et al.).However, because the antigen binding specificity is mainly due to theCDR regions, it has been possible, by using DNA recombinant techniques,to engineer chimeric antibodies that exploit the binding specificity ofmurine CDR mounted on the framework regions of human antibodies. Thespecificity of such antibodies turns out to be the same as for themurine antibody. The present invention is composed of amino acid andnucleotide sequences of the 3 CDR regions of the variable part of thelight chain and of the 3 CDR regions of the variable part of the heavychains (SEQ ID NO:7, 8, 9 respectively). Therefore are included in therange of the particular embodiment of the present invention allantibodies generated by “grafting” the CDR regions or at least the thirdCDR, corresponding to the SEQ ID NO:9, in other antibody supportstructure or in antibody-like support structures. Examples of the latterare “minibodies” in which the CDR of the invention arethree-dimensionally positioned in a way to maintain C5 bindingspecificity.

In agreement with this aspect, the present invention comprises in theessence, whichever recombinant human antibody able to recognise theepitope comprising the cleavage region of C5 convertase on activatedfactor C5. The recombinant human antibody is preferably characterised bythe fact that it comprises as CDR regions at least three of the aminoacid sequences identified in the sequence listing, like: SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9 or their conservative mutations.

Moreover the invention includes chimeric proteins comprising at leastone of the polypeptides corresponding to the SEQ ID NO:2, 4, or 6 orthose, obtainable by means of genetic engineering techniques, carryingconservative or non-conservative mutations and at least 95%, morepreferably 98% or even 99% homologous to the original sequences.Therefore the invention extends to polypeptides comprising at least oneof the antibody-specific amino acid sequences defined as SEQ ID NO:2, 4,6, 8, 10, 12, also when those are prepared in a form that differs fromthe canonical or natural form of antibodies. Also comprised in thepresent invention are embodiments of chimeric immunoglobulin comprisingat least one of the amino acid sequences identified as SEQ ID NO:2, 4 or6 or functional domains derived from such sequences in combination withthe amino acid sequence of Ig (immunoglobulin) heavy chain constantregions or subdomains of these regions (i.e. CH₂ or CH₃ domains) derivedfor example from amino acid (aa) sequences known in the database (i.e.human Ig A heavy chain gene of 00220, human Ig G heavy chain gene AF237583, Mus musculus L274371 g gamma heavy chain, Rattus norvegicusheavy chain region M28671). Preferably the antibody is dimeric. Theinvention also comprises sequences selected among SEQ ID NO:2, 4 or 6,when they carry mutations aa substitution or deletions which do notalter the binding specificity of the antibody of the invention.

The invention obviously extends to the amino acid sequences of theSequence Listing also when they comprise additional peptides positionedat the C or N-terminus, as for instance the “tag” or “flag” sequenceswhich are useful for purification or immunological recognition of therecombinant antibody in its various forms or when they carry deletionsat the C- or N-terminus, but which do not alter their bindingspecificity.

A particularly preferred type of “tag” sequence is the poly-histidinetail, coded by the expression vector used in the present invention,which is expressed at the C-terminal end of sequence ID NO:6 in order tofacilitate affinity purification on a nickel column. Another sequencetag is the SV5 of SIV, which is added in order to facilitateimmunological recognition.

A further aspect of the invention is represented by all the nucleotidesequences resulting from degeneracy of the genetic code, characterisedby the fact that they code the scFv antibody with sequence ID NO:6, orthe VH heavy chain corresponding to SEQ ID NO:4, or the light chaincorresponding to SEQ ID NO:2, and the nucleotide sequences codingpolypeptides having at least 95% homology preferably 98% or 99% homologywith the SEQ ID NO:6, the SEQ ID NO:4, the SEQ ID NO:2, preferably withSEQ ID NO:4, or coding for their conservative variants. Therefore thenucleotide sequences identified with SEQ ID NO:1, 3, 5 of the SequenceListing, or nucleotide sequences comprising such sequences, represent aparticularly preferred embodiment of the nucleotide sequences of theinvention.

Also included in the present invention are all the nucleotide sequencesobtained by means of “parsimonious mutagenesis” (Shier, R., et al.,1996, Gene 169: 147) or by means of other methods of random or directedmutagenesis of nucleotide sequences of the present invention, inparticular SEQ ID NO:1, 3, 5 (Marks, J. D., et al., 1992, J. Biol. Chem.267:16007) performed in order to improve some of properties ofantibodies, as for instance the affinity, while maintaining bindingspecificity for the C5 cleavage site. Moreover the present inventionincludes all scFv antibodies in which the sequence coding the VIIregion, corresponding to SEQ ID NO:3, is maintained constant, while thesequence coding the VL region of antibody TS-A12/22, corresponding toSEQ ID NO:1, is replaced by “chain shuffling”, for example usingcollections (“libraries”) of VL human regions.

The nucleotide sequences object of the present invention are cloned invectors suitable for their amplification, further mutagenesis ormodification or expression. Therefore, the vectors containing at leastone of the nucleotide sequences corresponding to SEQ ID NO:1, 3, 5, 7,9, 11 as in the sequence listing represent a further aspect of theinvention. These are preferably used for preparation of recombinantantibodies or of chimeric proteins in a suitable host and followingmethods known in the art.

In accordance to a preferred embodiment of the present invention, therecombinant antibodies are preferably cloned and expressed inprokaryotic hosts: particularly preferred is E. coli, but otherprokaryotic hosts can also be used, such as B. subtilis, P. pastoris, K.Lactis, or eukaryotic cells of plant or animal origin. Expressionvectors containing such nucleotide sequences are optimised forexpression in each of these hosts, by insertion of suitable regulatoryregions, promoters, transcriptional terminators or activators, orreplication origin. A particularly preferred expression vector accordingto the present invention is represented by a periplasmic expressionvector for E. coli, in particular the vector pDAN5 (Sblattero, D. andBradbury A., 2000, Nat. Biotechnol. 18:75).

The antibody or the chimeric proteins having the specificity describedin the present invention inhibit C5 conversion to its biologicallyactive products. In particular, by blocking C5b formation they blockformation of the terminal complex C (TCC) that leads to formation of theMAC (Membrane Attack Complex) which is capable of determining massivecellular lysis and significant tissue damage. Moreover, by inhibitingC5a formation they also inhibit the chemiotactic activity of C5a forPolymorphonuclear Leukocytes of and monocytes, that upon stimulationproduce either cytokines such as IL-1, IL-6, IL-8, or other importantinflammatory mediators such Serin-elastases, peroxidases, etc.

The C5a chemotactic activity and the cytolytic activity of MAC, toformation of which C5b participates, are considered the main causes ofinduced tissue damage in several inflammatory diseases. For instance,elevated levels of C5a fragment and of TCC have been found in thesynovial fluid of patients with rheumatoid arthritis or in thecerebrospinal fluid of patients with several diseases of the centralnervous system.

Therefore a further aspect of the invention relates to therapeutic useof recombinant anti-C5 antibodies of the invention, preferably human,carrying the ability of inhibiting the conversion of the C5 component toC5a and C5b, in one of the forms described in the invention: scFv, VHand/or VL alone or in combination with constant regions of Ig,preferably of human, rat or mouse origin, chimeric proteins or singlevariable regions and their conservative mutations, isotypic variantsetc. This aspect also relates to therapeutic use of their preferredembodiments composed of the amino acid sequences SEQ ID NO:2, 4, 6 ofthe Sequence Listing.

In accordance to the invention, the antibodies block conversion of C5 bythe C5 convertase. This enzyme can be activated by the classical pathwayinitiated by the C1q complement component triggered by antigen-antibodycomplexes or of IgG or IgM aggregates, or by the alternative activationpathway initiated by natural substances such as lectins, bacteria oryeast cell walls, or some snake venoms or nephritic factors. Thereforeit has been assessed whether antibodies or proteins of the inventionselectively block one or the other of the two complement activationpathways. Based on in vitro experiments performed by hemolytic assay onsheep erythrocytes (SRBC: Sheep Red Blood Cells) or on rabbiterythrocytes (RBC), the antibodies and proteins according to the presentinvention inhibit both classical and alternative complement activationpathways. An advantage provided by the use of antibodies or proteinshaving specificity for the C5 component in accordance to the presentinvention is that the initial complement components remain available forother functions of the complement system such as opsonization andclearance of immuno-complexes. This therapeutic approach turns out to beadvantageous over a potential block at the level of the C3 componentthat precedes the action of the C5 component. A block at the level ofthe C3 component would lead to a total block of the complement cascadeand of its functions in opsonization and clearance of immunocomplexes.

Therefore, in a particularly preferred embodiment, the antibodies or theproteins of the invention, in one of the described embodiments or inthose obviously derivable from them, are used for pharmaceuticalpreparations for treatment of diseases caused or accompanied byhyper-activation of the complement system. The invention extends the useof antibodies and proteins of the invention, and their coding nucleotidesequences, to the diagnostic field, in the area of diagnosis ofdisorders characterised or accompanied by uncontrolled activation of thecomplement system, in particular of the C5 component or of itsbiologically active fragments.

More preferably, polypeptides and antibodies of the invention are usedfor treatment or prevention of diseases caused or accompanied bycytotoxic and pro-inflammatory action of the terminal C complex(Terminal C Complex TCC), where in particular such diseases comprisechronic inflammation, and in particular rheumatoid arthritis,glomerulonephritis, multiple sclerosis, demyelinating peripheralneuropathies, atherosclerosis or some autoimmune disorders.

Moreover, experimental tests have demonstrated that anti-C5 polypeptidesand antibodies of the invention are therapeutically useful in bothtreatment and prevention of acute inflammatory pathologies induced oraccompanied by massive activation of the C5 component, which actsthrough the chemotactic activity of the C5a fragment, as well as throughthe TCC activity initiated by the C5b fragment. For instance, acutepathologies are represented by bacterial sepsis, tissue damage, forexample damage of the myocardium, of the central nervous, damages due totransplantation or caused by ischaemia and re-perfusion after ischaemia.

Moreover, a further aspect of the invention concerns the therapeutic useof nucleotide sequences coding antibodies or proteins of the invention,or the vectors containing them. Those are preferably used in somaticgene therapy of diseases induced or accompanied by massive activation ofthe C5 component, by uncontrolled activation of the complement system,by excess production of C5a and C5b fragments, by excess production ofcomplement components C5 to C9, as in rheumatoid arthritis or in someautoimmune disorders. Being subjected to individual variability andgeneral health status, it is not easy to quantify a higher than normallevel of activation of the complement system and of conversion of theactivated C5 component to its biologically active fragments. Thereforeabnormal levels are pragmatically defined as those that are able tocause an acute or chronic pathological state.

In vivo and in vitro production of antibodies of the invention intransgenic animals, obtained by genetic manipulation of non-humanmammals using at least one of the nucleotide sequences described in thepresent invention by methods known to the expert of the field, is alsocomprised within the scope of the present invention. This represents auseful application to the study of genetic diseases characterised byinsufficient production of C5a and C5b or by hyperactivation ofcomplement components from C5 to C9.

Antibodies and proteins of the invention and their coding nucleotidesequences can be prepared for therapeutic use in the form ofpharmaceutical compounds in combination with suitable excipients and/ordiluents preferably for parenteral administration.

Moreover, the antibodies of the invention allow the preparation ofdiagnostic, therapeutic or research kits, where in such kits comprise atleast one of the antibodies or of variable chains described in theinvention, corresponding to sequences SEQ ID NO:2, 4, 6, or theirhomologous variants or fragments generated for diagnosis or prognosis ofdiseases characterised by hyperactivation of the C5 component or theirencoding DNA sequences. The nucleotide sequences of the inventioncorresponding to sequences SEQ ID NO:1, 3, 5 and their homologoussequences, are preferably used to prepare kit for transfection ofeukaryotic cells.

The present invention also comprises the realization of a kit forselecting compounds that interfere or modulate the cleavage of the alphachain of the C5 complement component in its biologically activefragments. Realization of such kit involves the use of the polypeptidesof the invention, preferably the VH chain (SEQ ID NO:4) or the scFvchain (SEQ ID NO:6) and at least one of the following polypeptides: C5complement component and its alpha chain, or a peptide comprising thecleavage site for C5 convertase, preferably corresponding to sequenceSEQ ID NO:15. The compounds can be identified, as an example, by meansof a competitive assay detecting the inhibition of peptide-antibodybinding using at least one of the sequences of the invention. Theantibodies and polypeptides of the invention, independently from thefact that they originate from a human antibody library, arecharacterised by the ability to recognise the C5 complement component atthe level of an epitope present in the alpha chain of the C5 componentand comprising the cleavage region of C5 convertase. The bindingspecificity of the antibody of the invention for the epitope comprisingthe cleavage site for C5 convertase is situated between glycine 733 andarginine 734 in human C5, according to the SwissProt Data Base numbering(SEQ ID P01031). This epitope could be differently located in C5 ofnon-human mammalian species such as rat, mouse, rabbit; however it willbe considered as having the same specificity, would it have the effectto block conversion of the respective C5 to C5a and C5b.

Therefore, according to a further aspect, the invention relates to theuse of anti-C5 antibodies of the invention for setting up animalexperimental models of diseases induced or accompanied byhyper-activation of the complement system and in particular of the C5component.

In accordance with a further aspect, the invention comprises also thepeptide of complement factor C5 pertaining to mammalian species, andpreferably human, that comprises the cleavage site for C5 convertase.This peptide is preferably that comprising the region corresponding toamino acids 731-740 of the human mature protein. Even more preferablythe peptide has sequence KDMQLGR↓LHMKTLLPVSK (SEQ ID NO:15),corresponding to amino acid sequence 727-744 of the human protein. Thepeptides according to this last aspect of the invention are used forpreparation of medicaments, for example vaccines, or as immunogen toprepare antisera for prevention and treatment of pathological conditionsinvolving uncontrolled activation of the C5 complement component, suchas rheumatoid arthritis or some autoimmune disorders.

In accordance with this further aspect, the invention includes also theuse of a peptide comprising the region corresponding to amino acidsequence 731-740 of the human mature protein, or of a peptide sharing atleast 80% homology with such region, more preferably at least 90% or 95%or 98% homology including the corresponding regions of the C5 componentof non-human mammals. Even more preferably, this aspect of the inventionincludes the use of a peptide having sequence KDMQLGR↓LHMKTLLPVSK (SEQID NO:15) (derived from the human protein) or of a peptide having atleast 80%, more preferably at least 90% or 95% or 98% homology to suchpeptide, together with or in alternative to anti-C5 recombinantantibodies, in particular those comprising the variable region of the VHheavy chain, corresponding to SEQ ID NO:4, or the scFV antibody,corresponding to SEQ ID NO:6, to be used for selection of drugs andantibodies, even recombinant, capable of inhibiting conversion ofactivated C5 factor to its biologically active fragments C5a and C5b.

EXPERIMENTAL SECTION Materials

Antibody library. The antibody library used, having a complexity of7×10⁹, was derived from human non-immune (naive) peripheral lymphocytes.The construction of this library is described in Sheets, M. D., et al.,1998, Proc. Natl. Acad. Sci. USA 95:6157.

Bacteria. Phage amplification was in E. coli strain DH5aF′ (F′/endA1hsdR17 (rK⁻mK⁺) supE44 thi-1 recA1 gyrA (Naf) relA1 D (lacZYA-argF) U169deoR (F80dlacD(lacZ)M15)). For scFv fragment preparation was used E.coli strain HB2151 (K12, ara (lac-pro), thi/F′ proA⁺B⁺, lackl^(q) ZM15).

Purified proteins. Purified components C4 to C9 were purchased fromQuidel (Saint Diego, Calif.) and the human recombinant C5a component waspurchased from SIGMA-ALDRICH® S.r.l. (Milan, Italy). C5 alpha and betasubunits were Obtained by incubating 50 μg of C5 diluted in 0.55 MTRIS-HCl pH 8.1 containing DTT (0.02 M) for 30 min at room temperature(RT), followed by treatment with iodoacetamide (0.12 M) for 1 hour atRT. Alpha and beta chains were purified by gel filtration on Superose 12(Pharmacia Biotech, Milan, Italy) in Fast Protein Liquid Chromatography(FPLC) and purity was assessed by SDS-PAGE in non-reducing conditions.

Sera. A serum lacking in the C5 complement component (C5D) was obtainedfrom a patient with meningococcal infection. C5 levels were belowdetection and haemolytic activity was lacking in this serum, and wasrestored by addition of exogenous of C5. Human serum obtained from blooddonors was used as a source of C5 factor.

Preparation of the intermediate EAC1-3b for hemolytic assay.

Sheep erythrocytes (SBRC: Sheep red blood cells) were sensitised withsub-agglutinating amounts of rabbit IgM (EA). The EAC1-3b intermediatefor hemolytic reaction was prepared by incubating antibody-sensitisederythrocytes (EA) with 1/10 diluted C5 depleted serum (C5D) in saltbuffer containing glucose and Veronal (GVBS). Incubation was performedfor 70 minutes at 37° C., followed by addition of suramine (Bayer, FRG)to block the degradation reaction, as described by Harrison, R. A., andP. J. Lachmann (Harrison, R. A., and P. J. Lachmann. 1986. Complementtechnology. In Handbook of Experimental Immunology. D. M. Weir, L. A.Herzemberg, C. Blackwell, and A. Herzemberg Leonore, eds. BlackwellScientific Publ, London.

Antisera. Two anti-C5a monoclonal antibodies (Oppermann et al.Complement Inflamm., 1991, 8:13) and a goat anti-C5 antiserum (Quidel,Saint Diego, Calif. U.S.A.).

Example 1 Amplification and Selection of Phage Library

Phages were obtained and amplified as described in Marks, J. D. et al.,1991, J. Mol. Biol. 222:581. The selection was performed in“immunotubes” (Nunc, Mascia Brunelli, MI, IT) coated with purified C5protein. Coating of “immunotubes” was obtained by incubation with a C5solution (10 μg/ml in PBS) overnight at 4° C. Phages were diluted in PBScontaining 2% non-fat dry milk (MPBS) and incubated in immuno-tubes for60 minutes at room temperature. After incubation, the immunotubes werewashed 20 times with PBS containing 0.1% Tween 20 (PBST) and 20 timeswith PBS. The phage particles bound to immunotubes were eluted with 1 mlof E. coli bacterial culture at a density of 0.5 OD₆₀₀ for 30 minutes at37° C. Ampicillin (75 μg/ml), helper phage and kanamycin (25 μg/ml) werethen added and the culture was grown overnight.

After a second selection cycle on C5 coated immunotubes, eluted E. colicells were amplified to extract phagemid DNA using methods known in theart. The extracted DNA was used as template for separate PCRamplifications of VH and VL regions, and subsequent assembly and cloningin pDAN5 vector (Sblattero, D., and Bradbury A., 2000, Nat. Biotechnol.18:75). The pDAN5 vector is a phagemid vector containing lox and His₆regions and the recognition region for the p27 SV5 protein of SIV virus,characterised by the fact that VH and VL regions inserted in the vectorare expressed in VL-VH order, with the light chain at the N-terminus.After transformation with the phagemid, E. coli cells were incubatedwith the helper phage and phage particles were used for a thirdselection cycle. After elution, clones were analysed for their abilityto bind C5 according to what is described in is Example 2.

Example 2 Isolation of Phage Particles with Binding Specificity for theC5 Antigen

The phage particles obtained after three cycles of C5 “panning” inimmunotubes (as described in Example 1), were used to infect E. colicells grown on solid medium. Single bacterial colonies were transferredin 96-well plates and the resulting phages were further tested by ELISAfor their ability to bind C5. The C5 antigen, at a concentration of 10μg/ml in 0.1 M bicarbonate buffer pH 9.6, was bound to the plates byovernight incubation at 4° C. After saturation of plates with MPBS (PBScontaining 2% non-fat dry milk), 50 μl of phage suspension were dilutedwith an identical volume of MPBS; a monoclonal antibody anti-pIII (M13protein) conjugated to HRP (Pharmacia Biotech) was then added. Positivebinding was revealed by addition of H₂O₂ and3,3′,5,5′-thetramethylbenzidine diclorate (SIGMA-ALDRICH®) as substrateand absorbance at 450 nm by spectrophotometric reading.

Positive clones by the ELISA assay were further selected on the basis ofthe difference of V region coding fragments. V regions were amplifiedusing specific primers as described in Marks et al. (cited work),cleaved with BstNI and electrophoretically separated on 2% agarose gel.Twelve clones were isolated that proved to be different from each otherbased on their electrophoretic pattern.

Example 3 Preparation of Soluble Single Chain ScFv Antibodies

Phage clones obtained as described in Example 2 were used to infect theE. coli strain HB2151 to obtain expression of scFv fragments in solubleform. Bacteria grown until O.D. 0.5 in 2XYT medium, containingampicillin, were induced with isopropil-β-tiogalattopiranoside andfurther grown for 5 hours. The periplasmic fraction containing scFvantibodies was prepared by incubation with B-PER reagent (Pierce,Celbio, MI, IT) for 20 minutes at RT, followed by centrifugation for 15minutes at 27000×g. The supernatant was dialyzed against PBS and singlechain antibodies containing the poly-histidine tail at the C-terminuswere purified by affinity chromatography on nickel Ni-NTA resin(QIAGEN®, MI, IT).

The binding capacity of purified single-chain antibodies for factor C5was verified by means of a solid-phase ELISA assay. This was analogousto that used for phage particles. Rather than the antibody directed atpIII of M13, the antibody used for detection was a monoclonal antibodyagainst peptide SV5-tag expressed at the C-terminus of single chainantibodies.

The protocol for the ELISA assay was the following: wells of a 96 wellmicrotiter plate were coated with antigen (purified C5, 250 ng) byovernight incubation at 4° C. in 0.1 M sodium bicarbonate buffer, pH 9.6and then washed with PBS 0.1% Tween 20 (PBST). Residual binding siteswere blocked with PBS containing 1% BSA for 1 hour at 37° C. Bacterialextracts or purified single chain scFv antibody (1 μg/ml) were thenincubated and revealed with anti-SV5 tag antibody (diluted 1/1000)followed by goat anti-mouse IgG antibody (diluted 1/1000) during 1 hourincubation at 37° C. The enzymatic reaction was developed withp-nitrophenilphosphate (SIGMA-ALDRICH®; 1 mg/ml) as the substrate in0.1M glycine buffer pH 10.4 containing, 1 mM MgCl₂ and 1 mM ZnCl₂, andadsorbace at 405 nm was read by the ELISA reader Titertek multiskan.

All purified scFv antibodies were found to be capable of binding factorC5 by the described ELISA assay.

Example 4 Determination of the Binding Affinity of scFv Ts-A12 Antibodyfor Factor C5 by Surface Plasmon Resonance (SPR)

The binding affinity of the Tsa-12 antibody, purified by means of FPLCon Superdex 75 (Pharmacia), for factor C5 was measured by means ofBIACORE™ 2000 (a system that generates unique data on the interactionsbetween proteins and other molecules). The microchip (CMS, Biacore) wasprepared by direct conjugation of the C5 antigen with amines (20 μg/mlin 10 mM Sodium Acetate pH 4.5). The final level of immobilisationturned out to be approximately 1000 RU (Resonance Units). Associationand dissociation of antibody molecules from the bio-chip were measuredusing an optical detection system (Surface Plasmon Resonance).

The analysis was carried out at 25° C., at a flow of 15 μl/min, using 4different scFv concentrations in the range comprised between 100-300 nM,in PBS, 0.005% P20 (Biacore). The binding curves were interpolated 1:1according to the model of Langmuir, using the dedicated software termedBIAevaluation (version 3.5) with correction for the mass transfer.Measurements performed on the equilibrium dissociation constant arereported in table 1.

TABLE 1 Rates of association and dissociation of scFV TS-A12 on purifiedC5 immobilized on chip. clone k_(on) (10⁵ s⁻¹ M⁻¹) k_(off) (10⁻³ s⁻¹)K_(D) (10⁻⁹ M)^(a) TSA-12 1.3 0.026 200 ^(a)The equilibrium constant wascalculated as K_(D) = k_(on) /k_(off)

As it is shown in table 1, the KD calculated for the TS-A12 antibodyturns out to be of 2×10⁻⁷ M (sub-micromolar affinity), as expected forantibodies derived from collections (libraries) of non-immuneantibodies.

Example 5 Increased Affinity of the TS-A12 Antibody by Means of “ChainShuffling”

In order to increase the affinity, the TS-A12 antibody was subjected tosubstitution (“shuffling”) of the VL light chains. For this purpose thephagemid DNA was cut with BssHII and SalI to excise the VL region. Thisregion was substituted with all the repertoire of VL chains of apreimmune library (Sblattero, D., and Bradbury A., 2000, Nat.Biotechnol. 18:75) that was prepared with same enzymes. The library wassubjected to three cycles of selection on the antigen, as described inexamples 1 and 2, until a series of antibodies specific for C5 wasobtained. Among those is TS-A12/22 having dissociation constant, asmeasured with BIACORE 2000 according to the method described in theprevious example, of 1.8×10⁻⁸ M, therefore with an increment of molaraffinity of approximately one order of magnitude.

Example 6 Characterisation of the Inhibition from the Antibodies scFvAntiC5 of the Conversion of C5 to C5a

The obtained anti-C5 scFv fragments were further characterised for theirability to inhibit the hemolytic activity of C5 or, in other words, toblock conversion of the C5 component to C5a. For this purpose, a smallamount of purified C5 component was incubated, for 30 minutes at RT,with purified scFv anti-C5 antibodies, or with an unrelated scFv(anti-gliadin), or with VBS (Veronal Buffer Saline) as control. Themixture was then added to sheep erythrocytes sensitised withsub-agglutinating amounts of rabbit IgM (EA) (see Harrison, R, A., andP. J. Lachmann. 1986. Complement technology. In Handbook of ExperimentalImmunology. D. M. Weir, L. A. Herzemberg, C. Blackwell, and A.Herzemberg Leon ore, eds. Blackwell Scientific Publ, London) coated withcomponents from C1 until C3b (EAC1-3b) that allow to reveal C5activation through the classical pathway. The erythrocyte suspension wasre-suspended in C5 depleted serum (C5D) and incubated for 30 minutes at37° C. At the end of this procedure, cell lysis was measured aspercentage of the control of total lysis in distilled H₂O (see FIG. 1,panel B). The inhibition of C5a and TCC production by the scFv TS-A12and TS-A12/22 was measured by means of ELISA using the monoclonalantibody 17/5 as the capture antibody and antibody G25/2 as detectionantibody according to what has been described in Opperman et al., 1991,Complement Inflamm. 8:13. The presence of TCC was measured usingantibody aE11 as the capture antibody and a biotinylated anti-C5antibody (SIGMA-ALDRICH®) followed by alkaline phosphatase-conjugatedstreptavidin, as described by Tedesco et al. (1997, J. Exp. Med.185:1619).

The results of the test are presented in FIG. 1, where it is shown thatantibodies TS-A12 and TS-A12/22 inhibit almost completely formation ofC5a (FIG. 1A) (classical pathway) and of TCC (FIG. 1B) whereas the otherisolated scFv are effective only partially or to a limited extent. Thepresence of VBS or of an unrelated scFv in the reaction mixture did notshow, as expected, any inhibitory effect.

Example 7 Determination of the Sequence of TS-A12/22 Antibody

The VH and VL fragment of positive clones was compared with knownantibody sequences published in the VBASE data bank. The VH heavy chainof TS-A12/22 antibody was found to be derived from the VH3/V-48 chainand the light chain from Vλ3/V2-14. The DNA sequence coding the scFVTS-A12/22 antibody is reported in the Sequence Listing as SEQ ID NO:5,and the derived amino acid sequence as SEQ ID NO:6.

Example 8 Mapping of the Recognition Site of the TS-A12122 Antibody onthe C5 Molecule

It was first characterised, by means of the Western-blotting technique,whether the TS-A12/22 antibody recognises the alpha or beta of subunitof the C5 complement component prepared as described in Materials.Briefly the two subunits were electrophoresed in separate wells andtransferred on nitrocellulose membranes. A solution containing Tris 50mM pH 7.6, 0.5 M NaCl and 4% skimmed powder milk was used to blocknon-specific sites for 1 hour at 37° C. Membranes were revealed byincubation with a suitable dilution of the TS-A12/22 antibody for 1 hourat 37° C., followed by incubation with a secondary antibody labeled withalkaline phosphatase or with streptavidin conjugated with alkalinephosphatase (SIGMA-ALDRICH®). The enzymatic reaction was developed withblue tetrazolium and 5-bromo-4-chlorine-3-indolil phosphate(SIGMA-ALDRICH®, 0.30 mg/ml) diluted in 0.1 M Tris-HCl pH 9.5 containing0.1 M NaCl and 5 mM MgCl₂. Rainbow RPN 756 (AMERSHAM™ Italy (a trademarkof General Electric Healthcare Limited)) were used as molecular weightmarkers. As it is shown in FIG. 2, the TS-A12/22 antibody of theinvention recognises the alpha sub-unit of the C5 factor, loaded in lane1, while does not recognise the beta sub-unit, loaded in lane 2.

Since the purified TS-A12/22 antibody also inhibited hemolytic activitydue to inhibition of the conversion of C5 to C5a, as demonstrated inexample 9, the hypothesis that the antibody might recognise as bindingsite the cleavage site for C5 convertase was then verified.

In order to verify this hypothesis, a peptide of 18 amino acids wassynthesised, with sequence: KDMQLGR↓LHMKTLLPVSK (P5A-18 also termed C5cscomprising SEQ ID NO:15). This peptide corresponds to region 727-744 ofthe mature protein and comprises the cleavage site of C5 convertase(indicated by the arrow) between glycine 733 and arginine 734 accordingto SwissProt numbering for human C5 (SEQ ID P01031). The peptide wasused in a competitive ELISA assay on the C5 protein on solid phase.Before binding to the solid phase, the TS-A12/22 antibody waspre-incubated with the P5A-18 peptide, corresponding to amino acids727-744 of the C5 component, or with an unrelated peptide of sequenceGEEIQIGHIPREDVDYHLYP (SEQ ID NO:16 of sequence listing) corresponding toa fibronectin derived peptide termed CS5.

In FIG. 3 are shown the results that were obtained: the P5A-18 peptideinhibited binding of the TS-A12/22 antibody to C5, and this inhibitionwas dose-dependent, ranging from 45% to 90% for peptide concentrationsof 200 ng and 800 ng/200 respectively. This result confirms that theTS-A12/22 antibody recognises just this region on the activated factorC5. The concentration values correspond to a peptide Ki of 1 μM, that isnot different from the K_(D) measured by SPR for the entire protein,according to what has been described in example 4.

As expected, both the unrelated peptide CS5 and the whole C5 proteinresulted to be ineffective in inhibiting the binding of TS-A12/22antibody to activated C5, even at the highest concentrations used, thusindicating that inhibition by peptide C5cs (P5a-18) was specific for thealpha chain of C5.

Example 9 Functional Characterisation of Antibody TS-A12/22. Inhibitionof Hemolytic Activity and TCC Activation

Having assessed the characteristics of the inhibition of the classicalactivation pathway of factor C5 by the TS-A12/22 antibody, it was thenverified whether there was inhibition also of the alternative pathway ofC5 activation, and at which level. The test was carried out using rabbiterythrocytes as the target cells, according to well-known methods.Briefly, increasing amounts of C5 were mixed with 600 ng of the scFv ofthe invention or with an unrelated scFv or with GVBS in which scFvantibodies have been solubilised, and then incubated for 15 minutes atroom temperature. In order to estimate the classical pathway ofcomplement activation, each mixture was added to a 1% suspension ofsheep erythrocytes sensitised with rabbit antibody and coated withcomplement component up to C3b, termed EAC1-3b. In order to estimate thealternative pathway of complement activation, each mixture was added toa 1% suspension of rabbit erythrocytes.

C5 depleted serum diluted 1/200 was added to each erythrocytesuspension. After incubation for 30 minutes at 37° C., the percentage oferythrocyte lysis was measured by comparison to a 100% value obtained bylysing erythrocytes with an equal volume of distilled of H₂O and to ablank consisting of erythrocytes re-suspended in GBVS.Spectrophotometric reading was performed at 412 nm and the results arereported in FIG. 4: inhibition of EAC1-3b lysis through the classicalpathway (A Panel); inhibition of lysis of rabbit erythrocytes throughthe alternative pathway (B Panel). The TS-A12/22 antibody inhibitsrabbit erythrocyte lysis by inhibiting C5 conversion similarly throughthe classical pathway and the alternative pathway.

Since the amino acid sequence corresponding to the cleavage site ofconvertase on the C5 alpha chain is conserved in several animal species,it was furthermore evaluated, by hemolytic assay, the ability ofTS-A12/22 antibody to inhibit conversion of C5 to C5a also in rabbit,mouse and rat sera. The TS-A12/22 antibody was capable of inhibiting thehemolytic activity present in the serum of all animal species tested,though with different efficiency. These results are reported in FIG. 5,showing that the efficiency of inhibition of rat serum was practicallysimilar to that of human serum, whereas the efficiency was higher on rator rabbit serum. It should be also considered that a higher amount ofrabbit serum was necessary to induce a percentage of lysis comparable tothat induced by human C5.

(The specificity of TS-A12/22 antibody was evaluated also on othercomponents of the complement cascade, as for instance C3 and C4, whichare structurally similar to C5, but cross-reactivity was not found).

Example 10 In Vivo Use of TS-A12/22 Antibody

In order to test the effect of the TS-A12/22 antibody in vivo, theinflux of PMN was measured in the rat joint injected with BSA, BSA andTS-A12/22 or BSA and unrelated antibody. It was thus demonstrated thatPMN chemotaxis, that is their number in the joint wash out liquid, wassignificantly reduced in presence of TS-A12/22 compared with ratstreated with BSA or with BSA and unrelated antibody (FIG. 6). Moreoverthe deposition of the C3 and C9 complement components was checked on ahistological section of the joint of the back paw of treated rats, byimmunofluorescence with specific antisera and fluorescein-conjugatedsecondary antibody (FIG. 7). It was found that C3 deposition remainedunchanged as result of the administration of BSA plus TS-A12/22 antibodyor of unrelated antibody, but the deposition of C9 was stronglyinhibited in presence of TS-A12/22. It was thus confirmed that theTS-A12/22 antibody inhibits the complement cascade at a stepintermediate between C3 and C9 and therefore at the level of theconversion of C5 to C5a+C5b.

Example 11 Dimerization of scFvTS-A12/22 Antibody

The sequence of the TS-A12/22 antibody was modified by the addition ofCH3 or CH2-CH3 domains to the C-terminal end and of an eukaryotic leadersequence to the N-terminal end.

These modifications were aimed at:

inducing scFv dimerization, by forming a structural complex, in order toincrease the valence from one to two and to increase the stability ofthe antibody (hereafter defined “minibody”). Based on the differenttypes of cloning, covalent bonds are not formed between subunits of thedimer in constructs comprising only CH3, while in those comprisingCH2-CH3 domains are formed two disulfide bridges between cysteines ofthe CH2 hinge region of the two monomers;

making possible the production of minibodies in mammalian cell cultureswith increments of the yield and absence of bacterial contaminants atthe end of extraction and purification procedures;

creating the conditions for an in vivo analysis of the biologicalactivity of the minibody in animal models, minimising the host immuneresponse by adding CH3 and CH2-CH3 domains from the same species as thetreated animal (mouse or rat).

The above described modifications have been performed in two steps.First the scFv was cloned into plasmid vector pUT (Li E, et al. 1997.Protein Eng. 10:731-6) conveniently modified in order to allow insertionof the leader sequence and of a human CH3 domain. The CH3 human domainwas then replaced with a series of constructs of domains from otherspecies, as reported below. Finally, a fragment of pUT vector comprisingthe minibody in various versions, was cloned in the commercial vectorpcDNA3 (Invitrogen), and was tested for expression in cultured cell.

For this, the pUT vector containing an unrelated scFv was modified byreplacing the recognition site for the restriction enzyme (RE) BspEI,located at the end of the VH chain, with the BssH2 site. The BspEIsubstitution was performed by inverse PCR done with the primers reportedin Table 2 (references A and B).

The mouse CH3 sequence present in pUT vector was then replaced with ananalogous human sequence amplified by use of reference primers 1 and 2.Primer 2 inserts in addition a SV5 tag sequence (recognised by mAB SV5),and the cloning sites SpeI, PvuI and EcoRI. cDNA from human Blymphocytes was used as template and insertion into pUT was performed byrestriction with the RE BssHII and PvuI and subsequent ligation ofvector and fragment.

As indicated in FIG. 8, Fc domains of human, mouse and rat antibodieswere inserted into the pUT vector that had been modified as described inthe previous point. The original sequences of CH2 and CH3 domains wereobtained from an NIH nucleotide database using accession numbers:

Homo sapiens J00220 (locus: IgA1 heavy chain gene)

Homo sapiens AF237583 (locus: IgG1 heavy chain gene)

Mus musculus: L27437 (locus: Immunoglobulin gamma heavy chain)

Rattus norvegicus M28671: (locus: RATIGG2B).

Primers reported in Tab. 2 and cDNA from B lymphocytes of thecorresponding species were used for PCR cloning. The cloning in pUT wasperformed by BssHII and SpeI restriction and ligation after removal ofthe previous sequence (human CH3).

The scFv sequence was cloned into a series of pUT vectors containing Fcdomains of different species described in the previous point, by meansof PCR with a mix of universal oligonucleotides for amplification ofknown human scFv (see Sblattero D. and Bradbury A, Immunotechnology,1998, 3:271-278, and reported in table 3), followed by digestion withApaLI and BssHII restriction enzymes (RE).

Cloning in pcDNA vector of the constructs obtained in pUT was done bydigestion with HindIII and EcoRI enzymes, selective recovery of thecorresponding fragment and cloning in pcDNA3 that was cut with the sameRE (see outline in FIG. 8).

TABLE 2 Primers used for amplification of the Indicated Fc regions (Ra =rat; Mo = mouse; Hu: human). Name Orient. Sequence A SEQ. ID No. 17PUT-ApaII sense 5′ATC CGA GTG CAC ACC TGT GGA GAG AAA GGC AAA G 3′ B SEQID No. 18 PUT-BsshII antisense 5′TCC TCA GCG CGC GGC TCT GGT GGC AGA CCGAAG G 3′ 1 SEQ. ID No. 19 HuGCH3-s sense

2 SEQ. ID No. 20 HuGCH3-a antisense

3 SEQ. ID No. 21 HuGCH2-s sense

TGC CCA 3′ 4 SEQ. ID No. 22 HuA-CH2-s sense

5 SEQ. ID No. 23 HuA-CH3a antisense

6 SEQ. ID No. 24 MoG-CH3-s sense

7 SEQ. ID No. 25 MoG-CH3-a antisense

8 SEQ. ID No. 26 MoGCH2-s sense

9 SEQ. ID No. 27 RaGCH3-s sense

10 SEQ. ID No. 28 RaGCH3-a antisense

11 SEQ. ID No. 29 RaGCH2-s sense

TABLE 3 Universal Oligonucleotides mix for amplification of scFv ofhuman origin Name Orientation Sequence VL1 sense caggt gtg cac tcg gacatc crg dtg acc cag tct SEQ. ID No. 30 VL2 sense caggt gtg cac tcg gatatt gtg wtg acn cag wct SEQ. ID No. 31 VL3 sense caggt gtg cac tcg cagcct gtg ctg car yc SEQ. ID No. 32 VL4 sense caggt gtg cac tcg tcc tatgwg ctg acw cag cca SEQ. ID No. 33 JH1 antisense gaccc gcg cgc gga gacrgt gac cag ggt SEQ. ID No. 34 JH2 antisense gaccc gcg cgc aga gac ggtgac crt kgt SEQ. ID No. 35

Example 12 Production and Validation of the TSA12/22-CH2-CH3 RatMinibody

pcDNA plasmid carrying the construct for the TSA12/22 antibody, in theversion containing CH2-CH3 domains, was used for in vitro transfectionof the HEK 293 cell line. After treatment with DNA and lipofectin, cellswere selected in presence of the antibiotic G418. After two weeks inculture, single cell clones were assayed for antibody production bydetection of the activity in the supernatant. One clone was chosen andexpanded for massive production of the TSA12/22-CH2-CH3 minibody.Purification of the minibody was carried out by chromatography.

Purified TSA12/22 antibody in the version containing the rat CH2-CH3domains (minibody TSA12/22 CH2CH3) was tested for its ability to inhibitthe classical complement activation pathway in an in vitro test,according to the modalities already described in the previous examples.In the diagram of FIG. 9 it is reported the percentage of inhibition oflysis of rabbit erythrocytes sensitized with IgM, mediated by complementin presence of fixed concentrations of scFv TSA12/22, of an unrelatedscFv, of GVBS buffered salt solution and of the minibody composed ofscFv TSA22/12 and rat CH2-CH3 domains.

The Tsa12/22-CH2-CH3 construct showed significantly better inhibitoryactivity than the corresponding scFv, as can be noticed in FIG. 9.

In vivo activity tests in the rat joint space were also performed.Arthritis was induced at time t=0 by intra-articular injection ofmethyl-BSA (bovine serum albumin) after the animal had been previouslyimmunized with methyl-BSA. Therapeutic treatments with the minibody wereperformed by intra-articular injection at t=0 and 6 days after inductionof arthritis. Efficiency of treatment was measured as number ofpolymorphonuclear leukocytes present in the intra-articular wash out andas reduction of joint swelling, as can be noticed in FIG. 10 where it isreported the course of PMN (polymorphonuclear leukocytes) recruited tothe rat joint space in response to BSA-induced inflammation. The lowerpercentage of cells in presence of TSA22/12 CH2CH3 minibody, at two timepoints considered, indicates a reduction of the inflammatory process.FIG. 11 reports the inhibitory effect of the TSA22/12 CH2CH3 minibody onthe extent of joint swelling caused by the inflammatory reaction. Themeasurements performed after 20 days assessed a therapeutic effect ofthe minibody also in the long term, either when it was administeredtogether with BSA or 6 days later.

CONCLUSIONS

ScFv antibodies, isolated as described in example 1, were found to beable to bind to factor C5, as assessed by ELISA assay and as expectedfrom the type of selection or “panning” of phage particles performed onC5. However not all ScFv antibodies were able to inhibit conversion ofC5 to C5a+C5b, and therefore the biological functions following theiractivation. Because it binds in proximity of the C5 convertase cleavagesite on the alpha chain of the activated C5 component and preventsproduction of C5a and C5b, the TSA12/22 antibody inhibits thechemotactic activity induced by the former and the hemolytic activitymediated by C5b through MAC formation. This inhibition operatesdownstream of the activation of the C3 component, therefore it isindependent from the type of complement activation pathway utilised(classical or alternative).

Moreover, the TSA12-22 antibody dimerized by means of rat CH2 and CH3domains (minibody) turned out to be particularly active in the long termtreatment.

1. A human antibody having specificity for a C5 alpha chain of a C5component of the complement system characterized in that it recognizes aregion corresponding to sequence 727-744 (SEQ ID NO: 15) of the C5component of human complement or a region having at least 80% homologythereto, wherein said antibody inhibits the conversion of the C5 alphachain to C5a and C5b, wherein a light chain of the antibody is a lambdachain or a kappa chain, and a variable region of a heavy chain is theVH3 region.
 2. The antibody according to claim 1 characterized in thatit is recombinantly produced.
 3. The antibody according to claim 2,characterized in that it is in the form of single chain (scFv)comprising one variable region of the light chain covalently joined toone variable region of the heavy chain.
 4. The antibody according toclaim 1, characterized in that it comprises at least one of the aminoacid sequences selected from the group consisting of: SEQ ID NO:2, 4,and
 6. 5. The antibody according to claim 4 having amino acid sequenceSEQ ID NO:
 6. 6. The antibody according to claim 4 characterized in thatit comprises both the amino acid sequences identified as SEQ ID NO: 2and SEQ ID NO: 4, or their conservative mutations.
 7. The antibodyaccording to claim 4 characterized in that it comprises at least one ofthe sequences selected from the group consisting of SEQ ID NO: 2, 4, and6 in combination with a sequence derived from an immunoglobulin heavychain constant region.
 8. The antibody according to claim 7characterized in that it is dimeric.
 9. A chimeric antibodycharacterized in that it comprises at least one of the sequencescorresponding to SEQ ID NO: 2, 4, 6, 8, or 12, wherein the antibodyrecognizes a region corresponding to sequence 727-744 (SEQ ID NO:15) ofthe C5 component of human complement or a region having at least 80%homology thereto.
 10. A pharmaceutical composition comprising as theactive principle the human antibody of claim
 1. 11. The compositionaccording to claim 10 for myocardium reperfusion.
 12. A compositioncomprising any one of the antibodies selected from the group consistingof: an antibody against the activated C5 component of the complementsystem which recognizes a polypeptide having at least 80% homology withthe peptide comprising the region corresponding to sequence 727-744 ofthe C5 component of human complement, said peptide having the sequenceKDMQLGR↓LHMKTLLPVSK (SEQ ID NO: 15) and wherein said antibody inhibitsthe conversion of the C5 alpha chain to C5a and C5b; an antibodycomprising an amino acid sequences selected from the group consistingof: SEQ ID NO: 2, 4, and 6, or each one of SEQ ID NO: 7, 8, and
 9. 13.The antibody according to claim 1, characterized by the lambda chainbeing Vλ3/V2-14.
 14. The antibody according to claim 1, characterized bythe kappa chain being Vκ4/DPK24.
 15. The antibody according to claim 1,characterized by the VH3 region being VH3/V-48.
 16. The human antibodyof claim 1 or the chimeric antibody of claim 9 further comprising apeptide tag positioned at the C or N-terminus of said antibody orchimeric antibody, wherein said tag does not alter binding specificitiesof said antibody or chimeric antibody.
 17. The antibody or the chimericantibody of claim 16, wherein said tag facilitates affinitypurification.
 18. The human antibody or the chimeric antibody of claim17, wherein said tag is a poly-histidine tag.